Bottom Line:
However, inhibition of arginase by another arginase inhibitor S-(2-boronoethyl)-L-cysteine (BEC) had no effects.Moreover, the inhibitory effect of L-norvaline was not reversed by the NOS inhibitor L-NAME and L-norvaline did not interfere with TNFalpha-induced activation of NF-kappaB, JNK, p38mapk, while it inhibited p70s6k (S6K1) activity.The arginase inhibitor L-norvaline exhibits anti-inflammatory effects independently of inhibition of arginase in human endothelial cells.

Results: The induction of the expression of endothelial VCAM-1, ICAM-1 and E-selectin by TNFalpha was concentration-dependently reduced by incubation of the endothelial cells with the arginase inhibitor L-norvaline. However, inhibition of arginase by another arginase inhibitor S-(2-boronoethyl)-L-cysteine (BEC) had no effects. To confirm the role of arginase-II (the prominent isoform expressed in HUVECs) in the inflammatory responses, adenoviral mediated siRNA silencing of arginase-II knocked down the arginase II protein level, but did not inhibit the up-regulation of the adhesion molecules. Moreover, the inhibitory effect of L-norvaline was not reversed by the NOS inhibitor L-NAME and L-norvaline did not interfere with TNFalpha-induced activation of NF-kappaB, JNK, p38mapk, while it inhibited p70s6k (S6K1) activity. Silencing S6K1 prevented up-regulation of E-selectin, but not that of VCAM-1 or ICAM-1 induced by TNFalpha.

Conclusion: The arginase inhibitor L-norvaline exhibits anti-inflammatory effects independently of inhibition of arginase in human endothelial cells. The anti-inflammatory properties of L-norvaline are partially attributable to its ability to inhibit S6K1.

Figure 4: Role of arginase II silencing on TNFα-induced expression of VCAM-1, ICAM-1, and E-selectin. HUVECs were transduced with recombinant adenovirus expressing shRNA against LacZ as control (lanes 1 and 5) or arginase II (lanes 2–4 and 6 – 8). On day 4 of post transduction, cells were serum-starved for 20 h followed by stimulation with TNFα (10 ng/ml, 4 hours) and extracted. The knocking down effects of various shRNA targeting sequences were assessed by immunoblotting as shown in panel A. Panel B shows the effects on expression of VCAM-1, ICAM-1, and E-selectin. Shown are representative blots from five independent experiments.

Mentions:
Next we investigated whether the anti-inflammatory effects of L-norvaline are dependent on arginase activity. For this purpose, the endothelial cells were either pre-treated with another arginase inhibitor BEC (Fig. 3) or transduced with recombinant adenovirus expressing shRNA targeting arginase-II (Fig. 4), the principle arginase isoform in HUVECs [18]. Our experiments showed that BEC at the concentration of 200 μmol/L exerted inhibitory effects on arginase activity (51.9 ± 7.0% inhibition) which was comparable to that of 20 mmol/L L-norvaline (52.4 ± 3.9% inhibition, n = 4). However, in contrast to L-norvaline, BEC was unable to affect TNFα-induced expression of any of the inflammatory molecules (Fig. 3, n = 5). Similarly, knocking down arginase-II by the two different shRNAs i.e. shRNA-ARG IIa and c (shRNA-ARG IIb had no effect and served as a 2nd control in addition to shRNA-LacZ) also had no effects (Fig. 4, lanes 6 and 8 vs. lanes 5 and 7, n = 5).

Figure 4: Role of arginase II silencing on TNFα-induced expression of VCAM-1, ICAM-1, and E-selectin. HUVECs were transduced with recombinant adenovirus expressing shRNA against LacZ as control (lanes 1 and 5) or arginase II (lanes 2–4 and 6 – 8). On day 4 of post transduction, cells were serum-starved for 20 h followed by stimulation with TNFα (10 ng/ml, 4 hours) and extracted. The knocking down effects of various shRNA targeting sequences were assessed by immunoblotting as shown in panel A. Panel B shows the effects on expression of VCAM-1, ICAM-1, and E-selectin. Shown are representative blots from five independent experiments.

Mentions:
Next we investigated whether the anti-inflammatory effects of L-norvaline are dependent on arginase activity. For this purpose, the endothelial cells were either pre-treated with another arginase inhibitor BEC (Fig. 3) or transduced with recombinant adenovirus expressing shRNA targeting arginase-II (Fig. 4), the principle arginase isoform in HUVECs [18]. Our experiments showed that BEC at the concentration of 200 μmol/L exerted inhibitory effects on arginase activity (51.9 ± 7.0% inhibition) which was comparable to that of 20 mmol/L L-norvaline (52.4 ± 3.9% inhibition, n = 4). However, in contrast to L-norvaline, BEC was unable to affect TNFα-induced expression of any of the inflammatory molecules (Fig. 3, n = 5). Similarly, knocking down arginase-II by the two different shRNAs i.e. shRNA-ARG IIa and c (shRNA-ARG IIb had no effect and served as a 2nd control in addition to shRNA-LacZ) also had no effects (Fig. 4, lanes 6 and 8 vs. lanes 5 and 7, n = 5).

Bottom Line:
However, inhibition of arginase by another arginase inhibitor S-(2-boronoethyl)-L-cysteine (BEC) had no effects.Moreover, the inhibitory effect of L-norvaline was not reversed by the NOS inhibitor L-NAME and L-norvaline did not interfere with TNFalpha-induced activation of NF-kappaB, JNK, p38mapk, while it inhibited p70s6k (S6K1) activity.The arginase inhibitor L-norvaline exhibits anti-inflammatory effects independently of inhibition of arginase in human endothelial cells.

Results: The induction of the expression of endothelial VCAM-1, ICAM-1 and E-selectin by TNFalpha was concentration-dependently reduced by incubation of the endothelial cells with the arginase inhibitor L-norvaline. However, inhibition of arginase by another arginase inhibitor S-(2-boronoethyl)-L-cysteine (BEC) had no effects. To confirm the role of arginase-II (the prominent isoform expressed in HUVECs) in the inflammatory responses, adenoviral mediated siRNA silencing of arginase-II knocked down the arginase II protein level, but did not inhibit the up-regulation of the adhesion molecules. Moreover, the inhibitory effect of L-norvaline was not reversed by the NOS inhibitor L-NAME and L-norvaline did not interfere with TNFalpha-induced activation of NF-kappaB, JNK, p38mapk, while it inhibited p70s6k (S6K1) activity. Silencing S6K1 prevented up-regulation of E-selectin, but not that of VCAM-1 or ICAM-1 induced by TNFalpha.

Conclusion: The arginase inhibitor L-norvaline exhibits anti-inflammatory effects independently of inhibition of arginase in human endothelial cells. The anti-inflammatory properties of L-norvaline are partially attributable to its ability to inhibit S6K1.